Thesis

Comparative genomics and epigenomics of Sporosarcina ureae

Sporosarcina ureae is an aerobic, motile, spore-forming Gram-positive cocci that was originally isolated in the early 20th century from soil enrichments with elevated levels of urea. The species is unique in that it is the only known spore-forming cocci, and is currently placed in a genus exclusively composed of bacilli. Current research has been focused on the biotech potential of the unique outer cell surface layer (S-layer), and the ability to efficiently convert urea into ammonia. Specifically, researchers are using organisms that hydrolyze urea in applications such as self-healing concrete, biofuel production, and more efficient means to make fertilizer. The goal of this study is to utilize Pacific Biosciences (PacBio) DNA sequencing technology to generate complete genome sequences and to investigate genetic and epigenetic variations between strains of S. ureae that differ in their spatial and temporal isolation. We have sequenced the first six complete genomes and methylomes of S. ureae. Genomes were assembled using PacBio SMRT Analysis (v2.3.0) and Geneious (Biomatters; v9.0.4) software programs, and annotated using the Prokaryote Genome Automatic Annotation Pipeline . The average S. ureae genome is 3.3 Mb in size, and contains an average 3160 CDS, 66 tRNAs and 8 rRNAs, while only one of the strains contains a plasmid (64 kb). Epigenetic analysis, using SMRT Analysis and REBASE (New England Biolabs), of the strains demonstrated evidence of several novel adenine and cytosine methylases present in S. ureae. Examination of the species requirement of 97% sequence identity across the 16S rRNA gene was met by all six strains. However, further analysis using in silico DNA-DNA hybridization (DDH), average nucleotide identity (ANI), and additional core- and pan-genome analysis demonstrated a highly divergent species or possibly some of the strains were a subspecies or new species. Further genetic analysis of the entire genus is needed to determine exactly how S. ureae, a spore-forming cocci, relates to the other spore-forming bacillus species in the genus Sporosarcina. Utilizing genomics, our analysis has begun to clarify the make up of the genus, and also found that there may be additional species of spore-forming cocci other than just S. ureae.

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